1. Field of the Invention
The present invention relates to an apparatus and a method for performing a continuous free flow electrophoresis process. More particularly, the present invention relates to an apparatus and a method for performing a continuous free flow electrophoresis procedure in which crescent distortion of the samples is reduced by providing for means to introduce hydrodynamic compensation within the electrophoresis chamber to minimize the crescent distortion effects.
2. Description of the Prior Art
Electrophoresis, in general, in the phenomenon of the migration of charged particles or ions in a liquid carrier medium under the influence of an electric field. This phenomenon can be used to separate small particles which, by reason of different surface chemical properties, exhibit different concentrations of surface charge in the given medium. Under the influence of the electrical field, the electrophoretic mobilities of the various classes of charged particles in the carrier medium will be different. A sample continuously introduced at some point into the sheet of liquid carrier medium (buffer) flows in a narrow band in the absence of a potential gradient; however, when the potential gradient is applied to the sheet of buffer, the sample particles are separated under the influence of the electrical field into various particle groups or components depending upon the electrophoretic mobility of the respective particles, the strength of the field, and the length of time that the particles remain in the field. Particles of similar mobility are concentrated in distinctive zones or bands which fan out from the point of sample introduction.
The present invention relates in particular to free flow continuous electrophoresis in which a buffer solution is made to flow freely in a uniform film or sheet through a chamber defined by two parallel enlongate plates. A sample is introduced into the buffer sheet at some point, and an electric potential gradient is applied across this flowing sheet perpendicular to the direction of buffer flow. The individual components within each sample then separate into narrow bands depending upon their respective electrophoretic mobilities and can be collected from the outlet end of the electrophoresis chamber through one or more of a plurality of small tubes disposed along a collection manifold at the outlet of the chamber.
However, almost invariably, a number of factors inherent in the electrophoresis process combine to introduce distortion into the various individually deflected sample streams. This distortion, when examined in a cross section perpendicular to the flow within the chamber, assumes a crescent shape and is, therefore, most commonly referred to as "crescent distortion." The only known method for compensation for the crescent distortion effect is to employ appropriate coatings to the interior chamber walls such that the zeta potential of these coatings will induce an electro-osmotic flow velocity along the walls of the chamber which is approximately equal in magnitude but opposite in direction to the electrophoretic velocity of the individual sample which is desired to be collected. A publication by Allen Strickler and Terry Sacks in "Preparative Biochemistry," Volume III, No. 3 (1973), pages 269 to 277, teaches that crescent shaped deformations of the flow cross section of a given fraction, resulting from the velocity profile of the buffer stream, can be compensated by appropriate setting of the zeta potential of the separation chamber walls so that, for this fraction, the sample stream can be given a well defined cross section transverse to the flow direction, and thus optimum separation efficiency can be obtained. There is also a U.S. patent to Allen Strickler, U.S. Pat. No. 3,758,395 (Sept. 11, 1973) entitled "Resolution And Symmetry Control In Continuous Free Flow Electrophoresis" which teaches a method for crescent distortion compensation by adjusting the effective zeta potential of the chamber walls to optimize resolution by providing each of the walls of the apparatus with areas spaced along the length thereof having different zeta potentials and by either mechanically or electrically adjusting the relative electro-osmotic contribution of each area. Other U.S. patents involving continuous flow electrophoresis devices include U.S. Pat. No. 3,458,427 (July 29, 1969) to Strickler, U.S. Pat. No. 3,663,395 (May 16, 1972) to Strickler, U.S. Pat. No. 3,655,541 (Apr. 11, 1972) to Strickler, and U.S. Pat. No. 4,061,560 (Dec. 6, 1977) to Hannig, et al.
U.S. Pat. No. 3,458,427 to Strickler (July 29, 1969) bears a superficial resemblance to the method and apparatus of the present invention and for this reason bears further comment. This patent discloses a continuous free flow electrophoresis apparatus which includes means for varying the discharge flow rate of one portion of the flowing electrolyte sheet carrying the component bands with respect to the discharge flow rate of a second portion of the electrolyte sheet to cause a shift in the electrolyte sheet in a lateral direction to bring the desired component band into alignment with a collecting device. The essence of this device is then to shift the entire flow within the chamber, without compensating whatsoever for the distortion effects, in order that the single band of particulate material desired to be collected can be shifted to a single, fixed collection port for removal from the electrophoresis chamber. Apparently the phenomenon of crescent distortion was either unknown or ignored at the time of this patent, since no mention of the phenomenon is made in this patent, nor is any method proposed for its compensation.
As was mentioned hereinbefore, the only method heretofore disclosed for the compensation of the crescent distortion effect in an electrophoresis procedure is to adjust the zeta potential of the walls of the electrophoresis chamber, thereby inducing an electro-osmotic flow velocity which will minimize the crescent distortion effect. However, this method is only effective for a sample of a specific electrophoretic mobility. Hence, if more than one sample stream is desired to be collected, each such stream having a necessarily different electrophoretic mobility, the crescent distortion may be minimized for one of the sample streams only, leaving the other sample streams subject to the effect of the crescent distortion phenomenon. Also, the methods proposed for adjusting the zeta potential of the chamber walls tend to be rather cumbersome and time consuming in their application, thereby limiting the use of the process to situations in which samples of only a relatively narrow range of mobilities will be separated. Consequently, the flexibility of the various electrophoresis chambers with the zeta potential compensation feature of the prior art is severely compromised because such chambers cannot be quickly adapted to handle a variety of different samples wherein the samples for each separation differ more than minimally in the range of electrophoretic mobilities of their individual components.